Neutralizing antibody responses to influenza infection and vaccination is highly variable in the population which can be explained by differences in health status, exposure history, and polymorphism of immune response genes. Since protection is also correlated with neutralizing antibody titers, any role that immunoglobulin germline gene polymorphism may play in this variability is important to establish, but has been difficult to investigate due to the use of numerous V, D and J genes in the genesis of immunoglobulins and the enormous combinatorial diversity that results from the pairing of rearranged VH and VL genes. However, the discovery of biased usage of the IGHV1-69 germline gene in antibodies targeting the stem domain of (HA) hemagglutinin (HV1-69-sBnAbs) has provided a unique opportunity to investigate any biological role that IGHV1-69 germline gene polymorphism might have in the anti-influenza antibody response. Importantly, HV1-69-sBnAbs mostly originate from the IGHV1-69 51p1 allele-like group that encodes a conserved CDR-H2 Phe54 that serves as major anchor residue with HA. Here we present an association analysis between IGHV1-69 polymorphism and the frequency of stem reactive Abs by using blood samples and clinical data obtained from an NIH H5VN04 vaccine trial. Our data shows a remarkable effect of IGHV1-69 locus polymorphism on the serum BnAb titers to the HA stem. Analyses of their expressed Ab repertoires have also provided evidence that copy number variation (CNV) also impacts IgM HV1-69-sBnAb responses. Results of heat map analysis show that there may be a shift in global IGHV germline gene usage in individuals based on the IGVH1-69 genotype. We propose that underlying genetic variation within the human IGHV1-69 locus might explain a significant fraction of inter- individual variation to influenza vaccination. Here we plan to enroll a cohort o 100 adult healthcare workers to participate in a study to examine the effects of IGHV1-69 polymorphism in shaping the BnAb response to seasonal influenza vaccination. Three blood specimens will be obtained from each subject, pre-vaccination, day 7 post vaccination (plasmablast peak) and at 32-56 days post-vaccination (memory B cell pool). In Aim 1 pre vaccination blood samples will be used to study the role IGHV1-69 locus polymorphism in shaping global IGHV germline gene utilization and in establishing the anti-influenza BnAb memory B cell pool. Genotyping will be by SNP and copy number variation (CNV) and phenotyping by serologic MN, HAI, HA binding studies, anti- HA single B cell cloning and antibodyome analysis. In Aim 2 heterogeneity at the IGHV1-69 locus will effect global germline gene utilization in the BnAb response to seasonal influenza vaccination. We propose that there is a direct genotypic-phenotypic relationship at the IGHV1-69 locus with regard to an individual's capacity to mount an anti-influenza BnAb response. This study is designed to provide the first evidence of this linkage. This new information on the role of IGHV polymorphism in influenza immunity can be used translationally to predict and monitor vaccine responses and to design vaccines to achieve universal responsiveness.